Development of a Tunable Collagen/Heparin Matrix for Investigating Mechanical Cytoskeletal Crosstalk

The cytoskeleton plays a crucial role in cellular architecture, directing both cellular shape and a variety of mechanically influenced tasks, such as cell division, sensing and adaptation, and motility. The cytoskeleton can dynamically alter its length and stiffness through sensing its environment, yielding specific cellular responses. Mechanical changes can also occur from the bottom up through changes in cytoskeletal crosslinking and motor protein activity, altering molecular-level stiffness and thus further complicating mechanochemical feedback loops. The relative influences of “top down” versus “bottom up” mechanics on cellular activity and to what extent they are integrated within cytoskeletal systems remain unclear. This inherent dynamic nature proves difficult to mimic in in vitro studies designed to probe protein interaction and function of the cytoskeleton. To understand how the local mechanical environment plays a role in cytoskeletal crosstalk, we integrated a mechanically tunable collagen/heparin matrix constructed with a layer-by-layer technique combined with actomyosin ensembles. The layers are characterized using a quartz crystal microbalance with dissipation monitoring (QCM-D), which can detect nanoscale changes in mass and viscoelastic properties. Using the QCM-D, we were able to show increases in thickness and viscoelasticity of the matrix with an increasing number of collagen/heparin bilayers. We have also demonstrated that actin will stably bind the matrix at multiple layer depths. The presence of these layers will mimic different cytoskeletal environment stiffnesses and be coupled with actomyosin ensembles to understand how changing the system-level mechanical environment will alter force generation dynamics of myosin II using optical trapping. Preliminary bundle experiments suggest that the presence of the collagen/heparin bilayers improves the stability of actin filaments and the rate of photobleaching seen on the microscope. This approach allows us to investigate the biophysical crosstalk mechanisms of actomyosin ensembles and elucidate the sensory force feedback loops that ultimately scale and influence large-scale cellular tasks.